Abstract

Accumulation of triosephosphates arising from high cytosolic glucose concentrations in hyperglycemia is the trigger for biochemical
dysfunction leading to the development of diabetic nephropathy—a common complication of diabetes associated with a high risk
of cardiovascular disease and mortality. Here we report that stimulation of the reductive pentosephosphate pathway by high-dose
therapy with thiamine and the thiamine monophosphate derivative benfotiamine countered the accumulation of triosephosphates
in experimental diabetes and inhibited the development of incipient nephropathy. High-dose thiamine and benfotiamine therapy
increased transketolase expression in renal glomeruli, increased the conversion of triosephosphates to ribose-5-phosphate,
and strongly inhibited the development of microalbuminuria. This was associated with decreased activation of protein kinase
C and decreased protein glycation and oxidative stress—three major pathways of biochemical dysfunction in hyperglycemia. Benfotiamine
also inhibited diabetes-induced hyperfiltration. This was achieved without change in elevated plasma glucose concentration
and glycated hemoglobin in the diabetic state. High-dose thiamine and benfotiamine therapy is a potential novel strategy for
the prevention of clinical diabetic nephropathy.

Nephropathy is a common complication of diabetes. It is characterized by the development of proteinuria, culminating in end-stage
renal disease with a particular high risk of cardiovascular morbidity and mortality (1). The initial stage of development of nephropathy, incipient nephropathy, is characterized by the onset of persistent microalbuminuria
and hyperfiltration. Hyperglycemia is a risk factor for the development of incipient nephropathy in both type 1 and type 2
diabetic subjects (2–4). Tight control of blood glucose (and blood pressure) decreases the risk of developing nephropathy but is not always achievable
because of limitations of current drug therapy (5).

High plasma glucose concentration leads to high cytosolic glucose concentration in renal endothelial cells and pericytes with
consequent biochemical dysfunction: activation of protein kinase Cβ, hexosamine, and polyol pathways; metabolic pseudohypoxia; mitochondrial dysfunction and oxidative stress; and accumulation
of advanced glycation end products (AGEs) (6). The link of high cytosolic glucose concentration to metabolic dysfunction was demonstrated by overexpression of the GLUT1
glucose transporter in renal mesangial cells that thereby acquired the characteristics of the diabetic phenotype, including
increased extracellular matrix protein synthesis and activation of the polyol pathway (7). Supporting studies of mesangial and endothelial cells in hyperglycemic culture have exemplified the key features of biochemical
dysfunction in hyperglycemia: the accumulation of triosephosphates (8), increased de novo synthesis of diacylglycerol and activation of protein kinase Cβ (9), oxidative stress linked to mitochondrial dysfunction [sustained by high glycerophosphate shuttle activity (10)], concomitant activation of the hexosamine pathway, and the accumulation of methylglyoxal with increased formation of AGEs
(11). Increased concentrations of triosephosphate glycolytic intermediates, glyceraldehyde-3-phosphate (GA3P), and dihydroxyacetonephosphate
(DHAP) is the trigger for these processes (8,10). A pharmacological strategy that countered triosephosphate accumulation in hyperglycemia would suppress multiple pathogenic
pathways and prevent the development of diabetic nephropathy. Activation of the reductive pentosephosphate pathway (PPP) by
high-dose thiamine therapy may achieve this by increasing transketolase (TK) activity and stimulating the conversion of GA3P
and fructose-6-phosphate (F6P) to ribose-5-phosphate (R5P) (Fig. 1). Supporting evidence for this intervention comes from studies showing the normalization of triosephosphates by activation
of the reductive PPP in human erythrocytes in hyperglycemic culture by high-dose thiamine (12) and the correction of delayed replication, activation of protein kinase C (PKC), increased hexosamine and AGE concentrations,
and oxidative stress in capillary and aortal endothelial cells in hyperglycemic culture by high-dose thiamine and S-benzoylthiamine
monophosphate (benfotiamine) (13,14). We investigated the effect of high-dose thiamine and benfotiamine therapy on the development of incipient nephropathy in
the streptozotocin (STZ)-induced diabetic rat model of diabetes with moderate insulin therapy. The minimum daily allowance
of thiamine for rats was 4 mg thiamine per kg diet (15). The concentration of thiamine in rat food in this study was 25.7 mg/kg, and hence normal control rats were not thiamine
restricted. The conventional indicator of thiamine sufficiency is the “thiamine effect,” the increase of TK activity with
a saturating amount of exogenous TPP in ex vivo assay. When this increase is ≥15% of TK activity in the presence of saturating
TPP, there is thiamine deficiency (16). In this study, incipient nephropathy developed over a 24-week period in the STZ diabetic rats, as judged by hyperfiltration
and microalbuminuria, and both high-dose thiamine and benfotiamine therapy prevented it.

RESEARCH DESIGN AND METHODS

STZ diabetic rats.

Male Sprague-Dawley rats, 250 g, were purchased from Charles River U.K. (Ramsgate, Kent, U.K.). They were kept two per cage
at 21°C, 50–80% humidity, with daily 14-h light cycle, and had free access to food and water. Diabetes was induced by intravenous
injection with 55 mg/kg STZ. Body weight and moderate hyperglycemia were stabilized by subcutaneous injection of 2 units Ultralente
insulin every 2 days. Thiamine and benfotiamine were given orally, mixed with the food, at high doses (7 and 70 mg/kg daily)
over 24 weeks to STZ diabetic and normal control rats. At 6-week intervals, venous blood samples (200 μl) were taken with
heparin anticoagulant and 24-h urine samples were collected. Plasma glucose concentration was determined by glucose oxidase
method, glycated hemoglobin HbA1 by boronate affinity chromatography, and urinary and plasma creatinine by colorimetric assay (diagnostic kits 510, 442, and
555; Sigma). Glomerular filtration rate (GFR) was deduced as (urinary creatinine/plasma creatinine) × urine volume. The development
of nephropathy was judged by the measurement of albuminuria and proteinuria. Urinary albumin was determined by SDS-PAGE (8%
gels) of urine by calibrated densitometry of the albumin (66.5 kDa) band after Coomassie blue staining. The concentration
of protein in the urine was determined by the Bradford method. Renal glomeruli were isolated by sieving of renal cortex tissue
through 80- to 200-μm sieves and washed with 0.85% saline at 4°C (17). Where required, glomeruli were homogenized in 10 mmol/l sodium phosphate buffer, pH 7.4 and 4°C, and membranes sedimented
by centrifugation (20,000g, 30 min, 4°C). All procedures were approved by the U.K. Home Office for work under the Animals (Scientific Procedures) Act
1986; project license 80/1481.

Metabolite and enzyme activity analysis.

Thiamine and thiamine monophosphate (TMP) were determined in food, plasma, and urine by reversed-phase high-performance liquid
chromatography with fluorimetric detection (18). TK activity was determined in glomerular extract and hemolysate by the method of Chamberlain et al. (19). For Western blotting, glomerular protein (150 μg/well) was separated by SDS-PAGE (10% gels), transferred to nitrocellulose
and blocked with 5% milk protein, and blotted with rabbit anti-rat TK IgG (supplied by Prof. F. Paoletti) and anti-rabbit
IgG-peroxidase conjugate, and blots were developed with enhanced chemiluminescence (AP Biotech, Amersham, U.K.).

RESULTS

Animal study groups in this investigation were as follows: normal controls (C), normal controls with high-dose (70 mg/kg per
day) thiamine (CT70) and benfotiamine (CB70) therapy, diabetic controls (DC), and diabetic animals given high-dose thiamine
(DT7 and DT70) and benfotiamine (DB7 and DB70) therapy (7 and 70 mg/kg per day). The mean food consumption of the rats is
shown in Table 1. Hence, at baseline, dietary thiamine exceeded the recommended daily allowance by ∼6-fold (C), 9-fold (D), 20-fold (DT7),
and 140-fold (CT70 and DT70) and in thiamine equivalents, 20-fold (DB7) and 100-fold (CB70 and DB70). Mean body weights in
the control groups increased from 268 and 235 g at baseline to 712 and 607 g after 24 weeks in thiamine and benfotiamine dosing
studies, respectively; this was not changed significantly by thiamine and benfotiamine therapy. Mean body weights in the diabetic
controls increased from 268 and 243 g at baseline to 351 and 318 g in thiamine and benfotiamine dosing studies, respectively;
similar increases in mean body weight were found for diabetic rats with high-dose thiamine and benfotiamine therapy (Table 1). The diabetic rats had the characteristics of the diabetic state in both studies: increased plasma glucose concentration
and increased glycated hemoglobin HbA1. At 24 weeks, in the thiamine study, glycemic indicator values were as follows: plasma glucose concentration, diabetic 25.1
± 4.3 versus control 6.0 ± 1.2 mmol/l (P < 0.001); and HbA1, diabetic 17.7 ± 1.6% versus control 9.0 ± 0.8% (P < 0.001). At 24 weeks in the benfotiamine study, glycemic indicator values were as follows: blood glucose concentration,
diabetic 31.8 ± 3.2 versus control 5.2 ± 1.4 mmol/l (P < 0.001); and HbA1, diabetic 19.7 ± 2.0% versus control 8.7 ± 0.7% (P < 0.001). These increased levels in the diabetic rats were not changed by high-dose thiamine and benfotiamine therapy except
for small decreases in HbA1 of CT70 at 18 weeks, CB70 at 12 weeks, and CB70 and DB70 at 24 weeks with respect to normal and diabetic controls, respectively
(Fig. 2).

Prevention of microalbuminuria and proteinuria by high-dose thiamine and benfotiamine.

STZ diabetic rats on insulin maintenance therapy develop microalbuminuria over 24 weeks (28). Microalbuminuria was evident in the diabetic rats from 6 to 24 weeks, increasing from normoalbuminuria (2.0–2.2 mg albumin/24
h) to 12−17 mg/24 h at 6 weeks with a further progressive increase to 19–33 mg/24 h at 24 weeks (P < 0.01). Thiamine and benfotiamine therapy inhibited the development of microalbuminuria by 70–80% with no dose-response
relationship evident (P < 0.01) except for benfotiamine at 6 weeks (Fig. 3A and B). Proteinuria showed similar changes, albeit with excreted amounts of protein, approximately fivefold higher than for intact
albumin, except that a dose-response was generally evident (except at week 18) for thiamine therapy but not for benfotiamine
therapy (except at 6 weeks) (Fig. 3C and D). Overall, there was no marked difference in potency between thiamine and benfotiamine.

Hyperfiltration developed in the diabetic controls. The increase in GFR in STZ diabetic rats was not decreased by thiamine,
but it was prevented by benfotiamine at 6–18 weeks. At 24 weeks of benfotiamine therapy, mild hyperfiltration had developed
in the diabetic rats (Fig. 4). A decline in GFR was not observed, as is typical of this experimental model.

Thiamine status, transketolase activity and expression, and activation of the reductive PPP.

We assessed the effect of high-dose thiamine and benfotiamine therapy on thiamine status of the study group rats by measuring
the plasma concentrations of thiamine and TMP; thiamine pyrophosphate (TPP) was not detectable in blood plasma. We discovered
the previously unrecognized characteristic of STZ diabetic rats, that they are thiamine deficient; the plasma thiamine concentration
was decreased 69% in diabetic rats in the thiamine dosing study and 48% in the benfotiamine dosing study, with respect to
normal controls. This was due to increased urinary excretion of thiamine. In the thiamine dosing study, urinary thiamine excretion
was 0.55 μmol/24 h in controls and 2.28 μmol/24 h in diabetic rats (P < 0.01) and in the benfotiamine dosing study, 0.35 μmol/24 h in controls and 2.27 μmol/24 h in diabetic rats (P < 0.01). There was an associated eightfold increase in the renal clearance of thiamine in STZ diabetic rats, with respect
to controls (thiamine study, 0.51 vs. 4.03 ml/min, P < 0.01; and benfotiamine study, 0.38 vs. 3.01 ml/min, P < 0.01). This was prevented by 70 mg/kg thiamine and 7 mg/kg benfotiamine but not by 7 mg/kg thiamine. High-dose thiamine
and benfotiamine therapy gave a dose-dependent increase in the plasma concentration of thiamine: 7 mg/kg thiamine normalized
and 70 mg/kg thiamine supranormalized plasma thiamine levels in diabetic rats (Fig. 5A); and both 7 and 70 mg/kg benfotiamine supranormalized plasma thiamine levels (Fig. 5B). Benfotiamine did not increase the plasma concentration of TMP significantly in control or STZ diabetic rats.

We determined the effect of increased availability of thiamine on glomerular TK activity. Diabetic rats had decreased TK activity
(thiamine study −29%, P < 0.05; benfotiamine study −24%, P < 0.001), which was normalized by thiamine and benfotiamine therapy (Fig. 5C and D). There was also a significant but small “thiamine effect” on glomerular TK activity in diabetic rats only: thiamine study
7.4 ± 3.3% and benfotiamine study 1.9 ± 1.0% (P < 0.05). Decrease in TPP cofactor availability, however, as assessed by the “thiamine effect,” did not account for most of
the decreased glomerular TK activity in diabetic rats and reversal of this by high-dose thiamine and benfotiamine therapy.
Rather, TK expression was decreased in diabetic rats and supranormalized in thiamine- and benfotiamine-treated control and
diabetic rats (Fig. 5E–G).

We next sought to assess if activation of the reductive PPP by thiamine and benfotiamine had countered biochemical dysfunction
associated with the development of diabetic nephropathy—activation of PKC, dicarbonyl stress, glycation, and oxidative stress.
Using an assay of in situ PKC activity, we found increased glomerular PKC activity in STZ diabetic rats, with respect to controls
(thiamine dosing study 38 ± 3% and benfotiamine dosing study 54 ± 6%). This was reversed in a dose-dependent manner by thiamine
and benfotiamine therapy (Fig. 6A and B). Specific PKC activities in cytosolic and membrane fractions of glomeruli stimulated with exogenous DAG were also determined.
Cytosolic and membrane fraction PKC activities were increased by approximately onefold in diabetic controls, and these increases
were reversed by 55 and 66% with 7 and 70 mg/kg thiamine (Fig. 6C and E). Similar effects were found for benfotiamine therapy (Fig. 6D and F). Neither high-dose thiamine nor benfotiamine completely reversed diabetes-induced increases in glomerular PKC activity.

Dicarbonyl compounds such as methylglyoxal, glyoxal, and 3-deoxyglucosone are implicated in carbonyl stress and in increased
formation of AGEs in diabetes and are linked to the development of diabetic nephropathy (29–31). Methylglyoxal concentration is increased by the degradation of abnormally high concentrations of GA3P and DHAP in cells
suffering cytosolic hyperglycemia (32). High-dose thiamine therapy was therefore expected to decrease the plasma concentration of methylglyoxal, and this was indeed
found. Glyoxal and 3-deoxyglucosone were also increased in the plasma of STZ diabetic rats, and surprisingly, the increased
plasma concentrations of these glycating agents were also decreased by thiamine (Fig. 7A).

We determined markers of glycation (FL and AGEs in glomerular protein) and oxidative stress (plasma protein thiols) in STZ
diabetic rats with high-dose thiamine and benfotiamine therapy. The methylglyoxal-derived AGEs MG-H1 and CEL were increased
approximately twofold in glomerular protein of diabetic rats and normalized in a dose-dependent manner by thiamine and benfotiamine
(Fig. 7B–E). There were smaller increases in CML in diabetic controls of borderline significance (thiamine study 0.50 ± 0.19 vs. 0.27
± 0.11 mmol/mol lysine and benfotiamine study 0.42 ± 0.17 vs. 0.26 ± 0.12 mmol/mol lysine; P < 0.05). These increases were partially reversed by thiamine and benfotiamine therapy. Glomerular protein FL was increased
in diabetic controls but was not decreased by thiamine and benfotiamine therapy. The concentration of plasma thiols was measured
as a marker of oxidative stress. Plasma protein thiols were decreased in STZ diabetic rats by 30% of control levels in both
thiamine and benfotiamine dosing studies. Benfotiamine therapy at 50 mg/kg reversed this decrease significantly; thiamine
(7 and 70 mg/kg) and benfotiamine at 7 mg/kg did not (Fig. 7F and G).

DISCUSSION

Investigating the effect of high-dose thiamine and benfotiamine therapy on STZ diabetic rats to prevent the development of
nephropathy, we found that STZ diabetic rats had abnormally low plasma thiamine concentration and glomerular TK activity,
although they were not thiamine deficient by the “thiamine effect” criterion (16). Plasma thiamine was decreased in diabetic rats by increased renal clearance. This was associated with increased urinary
excretion of thiamine. Excessive diuresis of the diabetic rats (increased 14-fold) and decreased thiamine reabsorption in
renal tubules are probable causes. Thiamine in the glomerular filtrate is reabsorbed in the proximal tubules by a thiamine/H+ antiport activity (33). Decreased thiamine reabsorption may be due to structural damage in the proximal tubular cells or metabolic derangements
(glucosuria, intracellular acidosis, or other effects).

TK expression is decreased by thiamine deficiency (34), and this was found for the diabetic control rats in this study. This contributed to the decrease in glomerular TK activity
in diabetic rats. High-dose benfotiamine therapy increased plasma thiamine levels in both control and diabetic rats. It prompted
higher increases in plasma thiamine concentrations of normal control and diabetic rats with the 7-mg/kg dose than thiamine.
Both high-dose thiamine and benfotiamine normalized glomerular TK activity and reversed the decreased expression of TK in
diabetic rats. Recently, high-dose benfotiamine therapy (80 mg/kg daily) was found to also increase the activity of TK in
the retina of Wistar diabetic rats, but no decrease in TK was found in diabetic controls (35). Clinical diabetes is also associated with a mild thiamine deficiency. Two studies found 18 and 76%, respectively, of diabetic
subjects studied to have plasma thiamine concentrations lower than the normal range minimum (36,37). In the latter study, with moderate glycated hemoglobin (9% Hb), only 21% of diabetic patients had RBC TK activity higher
than the normal range minimum, and the mean thiamine effect was 17%, indicating borderline thiamine deficiency (37). Our study suggests that such thiamine deficiency may exacerbate the risk of developing nephropathy.

In diabetic rats, high-dose thiamine and benfotiamine therapy prevented the development of microalbuminuria and proteinuria.
There was no clear evidence of a dose-response relationship except for proteinuria for high-dose thiamine therapy. High-dose
thiamine and benfotiamine therefore prevented incipient nephropathy. Whether they also prevent the progression from incipient
to overt nephropathy remains to be investigated.

Activation of PKCβ in renal glomeruli has been linked to development of diabetic nephropathy. In renal glomeruli, activation of PKCβ stimulated the expression of vascular endothelial growth factor, transforming growth factor-β, and extracellular matrix components
such as type IV collagen (38). PKC activation in cytosolic hyperglycemia occurred via increased de novo synthesis of diacylglycerol from DHAP via glycerol-3-phosphate
and stepwise acylation. We found increased glomerular PKC activity in situ and specific activities in cytosolic and membrane
fractions of renal glomeruli of diabetic controls. Both high-dose thiamine and benfotiamine markedly suppressed PKC activation
to a similar extent. These changes reflect increased expression of glomerular PKC in STZ-induced diabetes (38), and suppression of this by high-dose thiamine repletion is a likely contributory factor to the prevention of incipient
nephropathy.

Protein glycation occurs via direct reaction of glucose with lysine and NH2-terminal amino groups of proteins to form FL and other fructosamines that may degrade to form AGEs, and also by the reaction
of α-oxoaldehydes with proteins to directly form lysine and arginine residue-derived AGEs (39). High-dose thiamine repletion prevented the diabetes-associated accumulation of methylglyoxal. It may do this by preventing
triosephosphate accumulation that leads to increased formation of methylglyoxal. The accumulation of glyoxal and 3-deoxyglucosone
was also decreased by high-dose thiamine repletion, however, probably by maintenance of high levels of NADPH and reduced glutathione
(GSH) in tissues to sustain their rapid metabolism by NADPH-dependent 3-deoxyglucosone reductase and the GSH-dependent glyoxalase
system, which also detoxifies methylglyoxal (40). We found that the AGEs MG-H1 and CEL were markedly increased, and CML increased moderately, in glomerular protein of STZ
diabetic rats; high-dose thiamine and benfotiamine both suppressed AGE accumulation to a similar extent. MG-H1, and similar
hydroimidazolones derived from 3-deoxygluocosone and glyoxal, were major AGEs found in glomerular protein. All were increased
in STZ diabetic rats, and the increases were reversed in a dose-dependent manner by thiamine and benfotiamine therapy (data
not shown). CML and hydroimidazolones are thought to bind specifically to AGE receptors (RAGEs) of glomerular endothelial
cells, pericytes, and podocytes (41). Activation of RAGE in renal glomeruli is implicated in growth factor expression, leading to mesangial expansion and glomerulosclerosis
in incipient nephropathy. Suppression of AGE accumulation may contribute to the prevention of incipient nephropathy by high-dose
thiamine repletion (42).

We examined plasma protein thiols as an indicator of oxidative stress. They were consistently decreased in the diabetic controls
of both thiamine and benfotiamine studies, but only treatment with 70 mg/kg benfotiamine prevented this decrease. Because
7 mg/kg benfotiamine and both doses of thiamine prevented the development of proteinuria, high-dose thiamine repletion prevented
incipient nephropathy in STZ diabetic rats without obligatory concomitant suppression of oxidative stress.

These observations suggest that high-dose thiamine repletion suppressed the development of incipient nephropathy in experimental
diabetes in which effects on the PKC, glycation, and oxidative stress pathways were involved. We found similar effects on
human mesangial cells in hyperglycemic culture in vitro (R.B.-J., N.K., N.A., S.B., P.J.T., unpublished data). The primary
intervention was the prevention of thiamine deficiency and induction of TK expression with consequent activation of the reductive
PPP shunt. It is remarkable that these effects were achieved by increasing the dietary availability of thiamine to diabetic
rats by as little as 20 times the minimum daily allowance, although this was sufficient to prevent thiamine deficiency. Thiamine
deficiency exacerbated the development of diabetic nephropathy. We therefore propose that clinical diabetic subjects should
avoid becoming thiamine deficient, even weakly so, and that high-dose thiamine repletion should be considered for therapy
to prevent the development of clinical diabetic nephropathy.

Acknowledgments

We thank the Juvenile Diabetes Research Fund and the Wellcome Trust (U.K.) for support for research. We also thank Professor
F. Paoletti, Department of Experimental Pathology and Oncology, University of Florence, Italy, for the gift of polyclonal
antibodies to rat transketolase.

The Diabetes Control and Complications Trial Research Group: The effect of intensive treatment of diabetes on the development
and progression of long-term complications in insulin-dependent diabetes mellitus.
N Engl J Med327
:977
–986,1993